Ampure rnacleanxp

Lung derived RNA samples were treated with RiboZero H/M/R rRNA (Illumina, San Diego, CA) depletion mix following a 40μl total volume; 4μL reaction buffer, 8μL probes, and 28μL sample. All incubation times followed the Ribo-Zero manual. After Ampure RNACleanXP (Beckman Coulter, Brea, CA) purification, the enriched RNA was eluted in 6μL of water. Following the Truseq Stranded mRNA Library Preparation Guide, Revision E., (Illumina, San Diego, CA), 5μL was added to 13μL of Elute-Frag-Prime Buffer and continued through second-strand cDNA. Library preparation continued with the adenylation of ends following the manufacturer’s recommendations. Final libraries were visualized on a BioAnalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA) and quantified using KAPA Library Quant Kit (Illumina) Universal qPCR Mix (Kapa Biosystems, Wilmington, MA) on a CFX96 Real-Time System (BioRad, Hercules, CA). Libraries were diluted to 2 nM stock, pooled together in equimolar concentrations and sequenced on an Illumina MiSeq using a Micro v2 sequencing kit at 2 × 150-bp. These data were used to make a new 2 nM pool so that virus coverage was normalized across samples. Subsequent sequencing was performed on an Illumina NextSeq 550 using a Mid Output v2.5 sequencing kit at 2 × 150-bp. Viral genome read depth coverage was greater than 100× for all samples.

We treated MERS-CoV samples with RiboZero H/M/R rRNA Depletion Mix (Illumina, https://www.illumina.com) according to the manufacturer’s instructions. After purification with Ampure RNACleanXP (Beckman Coulter, ttps://www.beckmancoulter.com), we eluted enriched RNA and assessed it on a BioAnalyzer RNA Pico Chip (Agilent Technologies, https://www.agilent.com). We prepared second-strand cDNA according to the Truseq Stranded mRNA Library Preparation Guide (Illumina). We treated samples with RiboShredder RNase Blend (https://www.cambio.co.uk).
We visualized final libraries on a BioAnalyzer DNA1000 Chip (Agilent Technologies), and quantified them by using a KAPA Library Quant Kit (Illumina) and a universal qPCR Mix (Kapa Biosystems, https://www.roche.com) on a CFX96 Real-Time System (Bio-Rad Laboratories, https://www.bio-rad.com). We pooled libraries together in equimolar concentrations and sequenced them using MiSeq (Illumina) with on-board cluster generation and 2 × 250 paired-end sequencing. The cluster density was at 454 k/mm²/lane, resulting in 8.7 million reads passing filter/run and an average 85% greater than the Q30 score.